Abstract:The abuse of antimicrobial agents has led to the rapid dissemination of multidrug resistant bacteria around the world. The multidrug resistant organisms (MDRO) and antibiotic resistance genes (ARGs) can be transferred among humans, animals and environments. Especially, ARGs can be transferred through a horizontal manner between the same species or different species of bacteria, making the growing problem of bacterial resistance and resistance mechanisms more complex. Moreover, the multidrug resistant organisms with complex resistance patterns have posed an increasing threat to our health. Therefore, the use of antibiotics must be tightly regulated.

Abstract:Carotenoids, as a group of over 700 valuable unsaturated terpene compounds classified as carotene and xanthophyll family, are endowed with powerful nutritional value. Phytoene dehydrogenase is the key rate-limiting enzyme in carotenoids biosynthesis pathway, involved in catalyzing the conversion from colorless hydrocarbon phytoene to other pigmented carotenoids, and plays an essential central regulation role. The function of phytoene dehydrogenases from different organisms exist diversity. CrtP, CrtQ and isomerase CrtH are essential for the formation of lycopene in most Cyanobacteria, whereas PDS, ZDS and isomerase Z-ISO, CrtISO are in charge of producing lycopene in most algae and plants. Nevertheless, there is only one CrtI-type for the formation of neurosporene, lycopene or dehydrolycopene in most bacteria and fungi. In this review, isolation, characterization, functional diversity, transcription regulatory mechanisms and phylogenetic analysis of phytoene dehydrogenase from different organisms are illustrated. This paper will provide insights into phytoene dehydrogenase and may facilitate the optimization of carotenoids production in genetic engineering strategy.

Abstract:We compared different methods used in single-cell isolation and whole genome amplification of microbial cells, and summarized research progresses in single-cell genomics (SCG) in studying environmental microbes. The studies of single cell and SCG have become one of the hot topics in life sciences in recent years. As a newly developed technology following microbial metagenomics, microbial SCG can efficiently explore large amounts of information of the uncultivable microbial genomics from environment. Generally, following steps need to be taken to obtain microbial SCG:isolation of single microbial cells, whole genome amplification of the single cells, amplicon sequencing, and data analysis. Microbial SCG can be widely used to explore new functional genes that are not detectable by metagenomics and other traditional methods, detect uncultured microbes with extremely low abundance, and study the life evolution of microbial cells.

Abstract:[Objective] The aim of this study was to isolate and characterize manganese (Ⅱ)-oxidizing bacteria from surrounding area of manganese mine.[Methods] Mn (Ⅱ)-oxidizing strains were isolated based on the ability to produce brown Mn oxides on agar plates. The presence of Mn oxides was confirmed by using the leucoberbelin blue (LBB) assay. The isolate was identified by morphological and physiological characterization and sequence analyses of 16S rRNA gene, gyrB gene and gyrA gene. The phylogenetic relationship between the isolated strain and reported Mn (Ⅱ) oxidizers was also analyzed. LBB assay was used to indicate the kinetics of Mn (Ⅱ) oxides formation. The Mn oxides morphology and chemical contents were determined by scanning electron microscope with energy dispersive spectrometer and X-ray diffraction.[Results] An isolate, named strain CP133, with high manganese oxidizing activity was obtained and identified as Bacillus cereus. There were some phylogenetic differences between strain CP133 and other Mn (Ⅱ)-oxidizing Bacillus species isolated from deep sea and soils. Strain CP133 produced amorphous manganese oxides that adhered to spore surface after the stationary phase of the cell culture. About 0.3 mmol/L manganese oxides was obtained after 12 days.[Conclusion] A Mn (Ⅱ)-oxidizing Bacillus cereus was successfully isolated. Our results suggest the diversity of Mn (Ⅱ)-oxidizing Bacillus species, and help understanding biogeochemical cycles in manganese mine and surrounding soils.

Abstract:[Objective] Different co-expression strategies to express leucine dehydrogenase and glucose dehydrogenase in E. coli were done to observe the effect of expression of different enzyme. A recombinant strain with two high enzyme activities was built for efficiently asymmetric synthesis of L-tert-leucine.[Methods] The leucine dehydrogenase (ldh) from Bacillus cereus and glucose dehydrogenase (gdh) from Bacillus sp. were co-expressed by three different strategies, including co-expressing two genes in single vector, co-expressing two genes in two vectors and expressing fusion protein. The catalytic efficiencies of recombinant strains with different enzyme activity ratio in different modes of biocatalyst were compared to produce L-tert-leucine from its corresponding α-keto acids.[Results] Different co-expression strategies displayed a slight impact on leucine dehydrogenase expression, whereas, a greater impact on glucose dehydrogenase. All the activity of leucine dehydrogenase was normally expressed, but the fusion proteins lost the activity of glucose dehydrogenase. Besides, the activity of glucose dehydrogenase was also totally inhibited when the 6-histidine tag was fused at C termini, which indicated the additional 6-histidine tag considerately depressed the glucose dehydrogenase activity. After optimization of expression, three recombinant strains exhibiting high enzyme activity and different enzyme activity ratio were used to synthesis L-tert-leucine in the mode of cell-free extracts and whole-cell. Result displayed a great influence on the catalytic efficiencies resulted from the mode of catalyst and enzyme activity. When the cell-free crude culture broth of E. coli BL21/pET28a-L-SD-AS-G coexpressing two genes in single vector was used as biocatalyst, 15 g/L cell loading and 0.1 mmol/L NAD⁺ were enough to completely transform 0.5 mol/L trimethylpyruvate into L-tert-leucine.[Conclusion] The recombinant strain with high activities of leucine dehydrogenase and glucose dehydrogenase was achieved by co-expressing two genes in single vector without histidine tag in E. coli and L-tert-leucine was efficiently produced with this recombinant strain.

Abstract:[Objective] We studied the contributions of four pigment biosynthetic genes to validamycin A yield, biomass accumulation, and the color of fermentation broth via individual gene deletions.[Methods] The deletion mutants were obtained via homologous recombination. The titer of validamycin A was detected by HPLC. The transcription of validamycin biosynthetic genes was quantified by qRT-PCR, and the growth was measured with dry cell weight.[Results] Compared with the parent strain, the deletion of DOPA melanin genes increased the validamycin A titer from 20.6 to 23.1 g/L (by 12%), whereas the deletion of type Ⅲ polyketide synthase melanin genes showed no effect. The inactivation of type Ⅱ polyketide synthase spore pigment genes and ochronotic pigment genes decreased validamycin A production by 11.7% and 17.2%, respectively. All these mutant strains had no significant change in transcriptional level and the color of supernatant.[Conclusion] Pigment biosynthetic gene deletions showed different effects on validamycin yield and biomass accumulation, and the deletion of DOPA melanin biosynthetic genes redirected the precursor flux and successfully increased the yield of validamycin A.

Abstract:[Objective] Biofilm plays an important role during the infection cycle of Vibrio cholerae. In this study, we try to demonstrate the role of VcDsbA in the biofilm formation of V. cholerae.[Methods] By making the VcDsbA inframe knock-out construct, the vcdsbA null mutant (ΔdsbA) strain was obtained. And the complemented strain (CΔdsbA) was constructed by transferring a plasmid-coded VcDsbA expressed under the control of arabinose to ΔdsbA strain. Crystal violet staining assay was used to analyze the biofilm formation in the wild-type (WT), ΔdsbA and CΔdsbA strains. V. cholerae strains containing msh promoter luxCDABE transcriptional fusion were used to analyze the transcriptional level.[Results] The ΔdsbA and CΔdsbA strains were constructed successfully. Biofilm formation analysis shows that the ability of biofilm formation of ΔdsbA was significantly reduced compared with WT, whereas CΔdsbA could form even stronger biofilm than WT does. Luminescence expression by Pmsh shows that VcDsbA enhanced msh expression. VcDsbA enhances the biofilm formation of V. cholerae by involving in the regulation of msh expression level. VcDsbA up-regulates msh expression probably through helping the folding of a msh expression activator.[Conclusion] VcDsbA plays an important role in the biofilm formation of V. chlerae, which makes the bacteria better survive in their living niche.

Abstract:[Objective] The molecular mechanism of antimicrobial peptide P7 against Escherichia coli was studied.[Methods] The binding mode between P7 and DNA was analyzed through fluorescence spectroscopy of P7 binding with E. coli genome DNA. The effects of P7 on E. coli cell cycle were determined through flow cytometry. Magnetic beads coupled with peptide were ussed to enrich peptide DNA-binding fragments, and PCR methods were used to analyze specific DNA to which P7 bound with. The influence of P7 on the gene expression levels of DNA replication and SOS damage and repair was analyzed through quantitative real-time PCR. The effects of P7 on E. coli DNA and RNA synthesis were analyzed according to the fluorescence spectra of nuclear stains.[Results] P7 intercalated into the base pairs of E. coli genomic DNA and then formed peptide-DNA complexes. As a result, the fluorescence intensity of the EB-DNA complex decreased. P7 could significantly increase the number of E. coli cells in phase S. The effect of P7 on normal E. coli cell cycle could significantly inhibit the DNA replication of E. coli. The binding of P7 with rnhA down-regulated the gene expression level by 2.24 times. The gene expression levels of ssb, dnaG, ligB, and rnhA that participated in E. coli DNA replication significantly decreased, and the gene expression levels of recA and recN in DNA damage and repair were significantly up-regulated under the effect of P7. P7 reduced E. coli DNA and RNA synthesis.[Conclusion] P7 also bound with rnhA. This binding resulted in DNA damage and inhibition of DNA replication of E. coli. P7 down-regulated the gene expression level of DNA replication, and the gene expression levels of DNA damage and repair were significantly up regulated. P7 reduced DNA and RNA synthesis of E. coli.

Abstract:[Objective] To establish a T7 promoter based reverse genetics system competent for the rescue of bovine parainfluenza virus type 3 (BPIV3).[Methods] We constructed three helper plasmids of px8δT-PT1-bPIV3-NP, px8δT-PT1-bPIV3-P and px8δT-PT1-bPIV3-L and one minigenome plasmid of pSC11-bPIV3-EGFP containing open reading frame (ORF) of the enhanced green fluorescent protein (EGFP) and cis-acting elements including BPIV3 leader region, gene start (GS), gene end (GE) and trailer region. All these plasmids are under the control of T7 promoter and identified by restriction endonuclease analysis. We rescued the pSC11-bPIV3-EGFP by two different methods. Then, we observed the fluorescence expression over time with fluorescence microscopy.[Results] We successfully constructed a reverse genetic system based 4 plasmids under the control of T7 promoter and finished the rescue operation to the minigenome of BPIV3.[Conclusion] This system can be further applied to investigate the function of BPIV3 genome by deletion and mutation of its genes.

Abstract:[Objective] Streptococcus equi subsp. zooepidemicus (GCS) is mainly used to produce hyaluronic acid (HA) in the industry. GCS secretes the hemolysis toxin (streptolysin S, SLS) that causes hemolysis in the host cells. Therefore, the safety of HA produced by GCS is concerned. We constructed an engineering strain, to produce commercial HA without SLS by knocking out sagA.[Method] The sagA of GCS was knocked out by the thermosensitive delivery vector system pJR700. The sagA mutant was identified through PCR with primers homologous to the flanking regions and SLS analysis. The yield of HA, HA molecular weight and virulence factors such as streptolysin Hylc, hyaluronate lyase, glyceraldehyde-3-phosphate dehydrogenase and cell surface proteins were determined by spectrophotometer and SDS-PAGE.[Result] We constructed successfully the in-frame deletion sagA mutant strain of GCS. In the sagA mutant, HA titer increased more than 30% than that of the wild type strain and no SLS hemolytic activity was detected. Compared to the wild type strain the sagA mutant decreased the quality of surface proteins, hemolytic Hylc activity and glyceraldehyde-3-phosphate dehydrogenase activity. The activities of hyaluronidase and cell were increased in the sagA mutant.[Conclusion] The sagA not only expressed hemolysis S but also regulated production of HA, the quality of surface proteins and activities of hyaluronidase, hemolysis Hylc and glyceraldehydes-3-phosphate dehydrogenase in Streptococcus equi subsp. zooepidemicus.

Abstract:[Objective] Under conventional cultivation conditions zebrafish harbors numerous microbes from the environment, leading to activation of its innate immune systems and interfering the results of relevant studies. We aimed to establish a germ-free zebrafish embryo model suitable for studies of host immune responses to infections.[Methods] A germ-free cultivation process including simple disinfection of the fertilized eggs and growth in a positive-pressured thermostatic isolator. Sterility testing of germ-free zebrafish embryos and water samples was done according to the national standards. The transcriptional level of TLRs, the mark genes indicating activation of the innate immune system, was detected by qPCR. Listeria monocytogenes was used as an infection model.[Results] The cultivation system and disinfection process could ensure germ-free status as shown by absence of microbes in zebrafish embryos and egg water. TLRs were barely detectable in zebrafish raised in the germ-free system, but highly induced in conventionally raised zebrafish or in germ-free zebrafish immersion-infected with pathogenic Listeria monocytogenes. The germ-free fish was sensitive to infection by L. monocytogene EGDe at a 100-CFU dose with 100% mortality in one week, while its isogenic mutants Δmpl and ΔplcB exhibited reduced death (70% and 40%, respectively). Macrophages were recruited around the intestine in EGDe immersion infected fish, but not in Δmpl and ΔplcB infected fish.[Conclusion] Zebrafish embryos produced by this simple process were free of microbes and could be used to study the innate immune responses and the pathogenesis of microbial pathogens.

Abstract:[Objective] To analyze adenovirus-mediated endosome lysis of T cells, we developed a novel approach based on pHrodo dextran (pH-sensitive fluorescent dye).[Methods] After incubating Jurkat cells (T cell leukemia) with serotype 5 adenovirus (Ad5) and pHrodo dextran, we determined the optimal incubation time and concentration of pHrodo dextran. To assess viral lysis of the endosome, we monitored the ratio changes of mean fluorescence intensity in different time points by laser scanning confocal microscopy.[Results] After incubating Jurkat cells with Ad5 and 80 μg/mL pHrodo dextran for 10 minutes, we observed the fluorescence intensity was significantly reduced at 30 minutes compared with that of endosomes at 0 minute. However, we found the mean fluorescence intensity was only slightly reduced by inhibiting V-ATPase with the bafilomycin A1 treatment.[Conclusion] The method based on pH-sensitive dye can be used to analyze the adenovirus-mediated endosome lysis of T cells.

Abstract:[Objective] This experiment was conducted to study different metabolic patterns of pig hindgut bacteria on aromatic amino acids by an in vitro fermentation method.[Methods] Ileum, cecum and colon chyme in Duroc, Landrace and Yorkshire goods hybridization pigs were taken as inoculum. The single aromatic amino acid concentration was kept 10 mmol/L in fermentation flask. Then the fermentation flask was incubated at 37℃ for 24 h. Gas production was measured at 4, 8, 12, 16 and 24 h, and samples of fermentation collected at 0 h and 24 h were used to measure ammonia nitrogen NH₃-N and microbial crude protein (MCP). Denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor and quantify the development of bacteria community in zymotic fluid.[Results] The concentrations of NH₃-N and MCP were significantly affected by aromatic amino acids and intestinal segments (P<0.01). Intestinal segments also affected gas production (GP) significantly (P0.01). NH₃-N, MCP and GP were affected by interaction of aromatic amino acids and intestinal segments. DGGE analysis showed bacteria of aromatic amino acids shared amount of bands together, especially similarity analysis of DGGE profile of Phe and Tyr in ileum, Tyr and Trp in colon were 87.9% and 80.5% separately. Shannon diversity indices analysis revealed that aromatic amino acids in cecum and colon varied significantly (P<0.05). Real-time PCR results showed that the quantity of total bacteria were affected by aromatic amino acids and intestinal segments significantly (P<0.05).[Conclusion] The potential as proportion of different aromatic amino acids are different. Compared with Trp and Phe, the diversity of bacteria utilizing Tyr in cecum or colon is low; compared with Tyr and Trp, a large number of Phe participated in synthesizing bacteria.The fermentation pattern of specific aromatic amino acids in different intestinal segment was unique. Compared with ileum and cecum, much more aromatic amino acids participated in the synthesis of bacteria in colon.

Abstract:[Objective] Single-chamber microbial electrolysis cell (SMEC), inoculated by granular sludge from upflow anaerobic sludge bed, can remove Ni(Ⅱ). Therefore, it is of great significance to explore the role microbial community to remove Ni(Ⅱ) in SMEC.[Methods] With sodium acetate as substrate, single-factor control method and Illumina high-throughput sequencing were applied to analyze Ni(Ⅱ) removal, as well as microbial community structure and dynamic characteristics in SMEC.[Results] Adsorption and microbial functions were the major mechanisms for removing Ni(Ⅱ). In mature single-chamber microbial fuel cell (SMFC), Geobacter sp. of Proteobacteria (91.42%) was dominant on the anode with an abundance of 76.25% whereas Niabella sp. of Bacteroidetes (47.99%) was dominant on the cathode with an abundance of 33.01%, followed by Ochrobactrum sp. of Poribacteria (45.74%) accounting for 10.08%. In SMEC modified from mature SMFC with 12.5 mg-Ni(Ⅱ)/L, the dominant bacteria turned from single Geobacter sp. to Geobacter sp. (41.56%) and Azospirillum sp. (5.97%) on the anode; the dominant bacteria on the cathode turned from Niabella sp. and Ochrobactrum sp. to Acetoanaerobium sp. (19.28%), Dokdonella sp. (16.48%) and Azospirillum sp. (9.49%).[Conclusion] Microbial populations in raw sludge were selectively acclimated in the Ni(Ⅱ) removal process of SMEC, and effective microbial communities of electrogenesis and nickel removal were built on the electrode to promote the removal of Ni(Ⅱ) in SMEC.

Abstract:[Objective] We assessed the phylogenetic relationship of Sugarcane streak mosaic virus (SCSMV) according to NIa sequences, to infer the prevalence and variation of SCSMV and to prevent and control this virus.[Methods] Leaf samples with mosaic symptom were collected from sugarcane-growing areas in Yunnan province and the Chinese national nursery of sugarcane germplasm resources (NNSGR). NIa sequences of SCSMV were determined by RT-PCR, and analyzed by Splits Tree, RDP, PhyML and DnaSP softwares, in aspect of phylogenetic, selection, and gene flow.[Results] We obtained 23 NIa sequences; clear recombination site was not found in NIa; a novel cluster formed by SCSMV Yunnan isolates determined here was found; strong purifying selection was found in NIa of SCSMV; and the gene flow of SCSMV subpopulations between sugarcane-growing areas in Yunnan province and the NNSGR was not frequent.[Conclusion] Similar with P1, HC-Pro and CP genes, SCSMV isolates could be divided into five clusters. NIa of SCSMV Yunnan isolates showed high genetic diversity and clear geographical distribution.

Abstract:[Objective] Sporolactobacillus inulinus, a typical homofermentative lactic acid bacterium, is an efficient D-lactic acid producer. Various environment factors affect the productivity of S. inulinus. Glucokinase, phosphofructokinase, pyruvate kinase and lactic dehydrogenase are the key enzymes of D-lactic acid production from glucose by S. inulinus. The characteristics of these enzymes are important in controlling and regulating the fermentation process. According to the genome bioinformatics analysis of S. inulinus CASD, three putative D-lactate dehydrogenases were identified, among which the bifunctional protein had been reported. In this study, we provided insights into the characteristics of the other two D-lactate dehydrogenase isozymes.[Methods] S. inulinus Y2-8 genome was used as the template to amplify D-lactate dehydrogenase gene (dldh) and D-isomer specific 2-hydroxyacid dehydrogenase gene (dhdh). The two recombinant strains E-pET-28a/dldh and E-pET-28a/dhdh were constructed for enzyme expression. Both recombinants DLDH and DHDH could convert pyruvic acid into D-lactic acid.[Results] Enzymes expressed by recombinant strains were purified by Ni-NTA chromatography. The apparent molecular mass of DLDH was approximately 37 kDa by SDS-PAGE analysis, and DLDH showed a high affinity to pyruvate with the K_m value of (0.58±0.04) mmol/L. The optimal reaction temperature and pH for DLDH was 35℃ and 6.5, respectively. The apparent molecular mass of DHDH was approximately 39 kDa, and the K_m of DHDH toward pyruvate was (1.70±0.08) mmol/L. The optimum catalysis temperature and pH of DHDH were 30℃ and 7.5, respectively.[Conclusion] According to the K_m and optimal reaction pH, DLDH was suggested as the main catalyst in formation D-lactic acid from pyruvate during the fermentation. The enzymatic properties would contribute to the regulation of the fermentation of S. inulinus.